Takahashi T, Ikai A, Takahashi K
Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo.
J Biol Chem. 1989 Jul 15;264(20):11565-71.
An enzyme hydrolyzing proline-beta-naphthylamide was purified to apparent homogeneity from porcine intestinal mucosa. The purified enzyme appears to consist of three identical subunit polypeptides with a molecular weight of about 58,000 each, associated noncovalently. The enzyme is a glycoprotein, and the subunit polypeptide contains 3 residues each of mannose and N-acetylglucosamine. A wide variety of peptidase substrates were tested for the enzyme, and the results showed that it hydrolyzes only aminopeptidase substrates, such as proline-beta-naphthylamide, glycine-beta-naphthylamide, leucine-beta-naphthylamide, and alanine-beta-naphthylamide. Among these substrates, proline-beta-naphthylamide is most efficiently hydrolyzed as judged by the kcat/Km value. The optimum pH for this substrate is around 9. The enzyme also hydrolyzes efficiently the ester substrates of these amino acids. No hydrolytic activity was observed for the peptide and protein substrates tested. The proline-beta-naphthylamidase activity was drastically inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, indicating that the enzyme is a serine hydrolase, whereas it was slightly inhibited by aminopeptidase inhibitors, such as amastatin, bestatin, and puromycin. No significant homology was found for the NH2-terminal sequence of 27 amino acid residues with any known protein sequences. From these results we conclude that the enzyme is a protein which has not been described before.
一种能水解脯氨酸-β-萘酰胺的酶从猪小肠黏膜中纯化至表观均一。纯化后的酶似乎由三个相同的亚基多肽组成，每个亚基多肽的分子量约为58,000，通过非共价键结合。该酶是一种糖蛋白，亚基多肽各自含有3个甘露糖残基和N-乙酰葡糖胺残基。针对该酶测试了多种肽酶底物，结果表明它仅水解氨肽酶底物，如脯氨酸-β-萘酰胺、甘氨酸-β-萘酰胺、亮氨酸-β-萘酰胺和丙氨酸-β-萘酰胺。在这些底物中，根据kcat/Km值判断，脯氨酸-β-萘酰胺被水解的效率最高。该底物的最适pH约为9。该酶也能有效水解这些氨基酸的酯底物。对于所测试的肽和蛋白质底物未观察到水解活性。脯氨酸-β-萘酰胺酶活性受到二异丙基氟磷酸酯、苯甲磺酰氟和L-1-对甲苯磺酰胺基-2-苯乙基氯甲基酮的强烈抑制，表明该酶是一种丝氨酸水解酶，而它受到氨肽酶抑制剂如抑氨肽酶、贝抑素和嘌呤霉素的轻微抑制。在27个氨基酸残基的NH2末端序列与任何已知蛋白质序列之间未发现显著同源性。根据这些结果我们得出结论，该酶是一种此前未被描述过的蛋白质。
